Methods:
This study followed the tenets of the Declaration of Helsinki involving human subjects and was approved by Institutional Review Board of our institute. After reaction with 0-100 mM of DVS, the AM samples were characterized by ninhydrin assays to determine the extent of cross-linking. The effects of cross-linker concentration on the mechanical and biodegradation behaviors of these biological tissues were also investigated. The interaction between cross-linked AM materials and human corneal epithelial cells was analyzed for indication of biocompatibility. After cell growth on various DVS cross-linked AM, the mitochondrial dehydrogenase activity and stemness gene expression were determined.

Results:
With increasing DVS concentration, the extent of cross-linking of chemically modified AM materials significantly increased. It indicates that the cross-linking efficiency is strongly dependent on the generation of cross-links (i.e., ether linkages) by collision between the AM collagen and DVS molecules. The Young’s modulus was positively correlated with the increased cross-linking degree of biological tissues. In addition, the samples treated with 100 mM of DVS had the lowest biodegradability after incubation in the buffer solutions containing collagenase. The results show that the amount of covalently incorporated DVS into the structure of AM collagen may play an important role in determining the stability of biological tissue. When the cross-linker concentration was below 50 mM, the DVS treated AM materials were compatible with human corneal epithelial cell cultures. The AM treated with higher cross-linker concentration could promote limbal epithelial cell proliferation and enhance stemness gene expression.

Conclusions:
The present study suggests that the cross-linker concentration may have a profound influence on the characteristics of DVS treated AM as a LEC scaffolding biomaterial.